N.H.R. Litjens, A.W. Langerak, A.C.J. van der List, M. Klepper, M. de Bie, Z. Azmani, A.T. den Dekker, R.W.W. Brouwer, M.G.H. Betjes, W.F.J. van IJcken
Wednesday 4 march 2020
0:00 - 0:00h at Toon Hermans Foyer
Parallel session: Postersessie 2 – Basale abstracts
Background: Single cell transcriptomics is a powerful tool for in-depth characterization of a heterogeneous pool of T cells and this assay can be combined with TRA/TRB T cell receptor (TCR-) repertoire analysis. The aim of this study was to evaluate the potential of this combined assay to eventually unravel mechanisms of donor-specific hypo-responsiveness in stable kidney transplant recipients 3-5 years after transplantation.
Methods: For this purpose, we carefully selected T cell lines with defined TRA/TRB clonotypes as well as clinical samples enriched for T cells, possessing a complex TCR repertoire. Low cell numbers of the different samples were dispensed in a Wafergen chip, messenger (m)RNA was isolated and cDNA prepared on the iCell 8 system. Two assays were performed on each single cell cDNA preparation, one for the TRA/TRB TCR-repertoire and one for the 5’ ends of transcripts. The 5’ libraries were sequenced on an Illumina HiSeq2500 sequencer, whereas the TRA/TRB TCR libraries were sequenced on an Illumina MiSeq system.
Results: On average 225 cells/sample were dispensed in a chip and 77% of the cells could be used for analysis of either TRA/TRB clonotype or transcriptome. The correct TRA/TRB clonotype was determined for on average 98.1% of the cells of the cell-lines. The TRA/TRB TCR-repertoire of the clinical samples was more complex than that of the cell-lines. The TRB clonotype distribution of the clinical samples was positively correlated to that obtained by flow cytometry-based approach (R=0.80). Transcriptome (5-prime) analysis revealed expression of on average 2351 unique genes/cell for the cell-lines and 770 genes/cell for the clinical samples.
Conclusions: In conclusion, combined single cell analysis of transcriptome and TRA/TRB clonotype can be applied to low frequent T cell populations. Complex TRA/TRB TCR-repertoires (clinical samples) can be distinguished from T cell lines having only one TRA/TRB clonotype. Moreover, samples with particular TRA/TRB clonotypes can now be analyzed in more depth in parallel with their transcriptome. This new technology now paves the way for in-depth analysis of pathways underlying donor-specific hypo-responsiveness in stable kidney transplant recipients through characterizing donor-reactive CD137-expressing T cells.